Ergosterol extraction: a comparison of methodologies

Ergosterol is a component of the cell membrane of mycorrhizal fungi and is frequently used to quantify their biomass. Arbuscular mycorrhizal (AM) fungi and ectomycorrhizal (ECM) fungi establish a symbiotic relationship with a respective host plant. Several methods are currently employed for quantification of ergosterol; however, these utilise a series of potentially hazardous chemicals with varying exposure times to the user. The present comparative study aims to ascertain the most reliable method to extract ergosterol whilst limiting hazard exposure to the user. Chloroform, cyclohexane, methanol and methanol hydroxide extraction protocols were applied to a total of 300 samples of root samples and a further 300 growth substrate samples across all protocols. Extracts were analysed via HPLC methodologies. Chromagraphic analysis showed chloroform-based extraction procedures produced a consistently higher concentration of ergosterol in both root and growth substrate samples. Methanol hydroxide, without the addition of cyclohexane, produced a very low concentration of ergosterol, with a reduction of quantified ergosterol of between 80 and 92 % compared to chloroform extractions. Hazard exposure was greatly reduced following the chloroform extraction protocol when compared with other extraction procedures.

The present study aims to compare different ergosterol extraction methods and comment on the degree of chemical safety each method presents to the user as well as the quantity of ergosterol extracted for reliable fungal biomass estimation. Ergosterol is analysed here via HPLC methodology.

METHODS
Plant growth of two ECM fungi-supporting species [English oak -Quercus robur (n=20), and roses -Rosa gallica (n=20)] and two AM fungi-supporting species [wheat -Triticum aestivum (n=20), oats -Avena sativa (n=20)] were grown under controlled conditions (20±2°C, 18±5 % humidity, 15 500 lumens) and grown in 50 % perlite and 50 % vermiculite as growth substrate with the addition of 50 g J Arthur Bowers multipurpose compost as a mycorrhizal inoculum. Watering was carried out once per week to a total volume of 100 ml, along with a source of liquid nutrient (BabyBio) applied to each plant every 4 weeks diluted as per the manufacturer's instructions. Plants were sampled at 6 months post-germination where a maximum mass of 15 g of growth substrate (n=300 per ergosterol extraction protocol per plant species) and 1 g of root tissue (n=300 per ergosterol extraction protocol per plant species) was taken and stored at −20 °C until ergosterol extraction could be performed.

Sample pre-treatment
A total of 2 g sampled rhizosphere growth substrate and 1 g root material was air dried at 25 °C for 48 h. Samples were determined to be dried after three consistent and consecutive weight measurements a minimum of 6 hs apart.

Ergosterol extraction non-alkaline (methanol) extraction protocol
A modified method of Millie-Lindblom et al. [9] was used for ergosterol extraction via methanol, as summarized in Fig. 1. Of both growth substrate and root samples, 300 mg was weighed into 50 ml centrifuge tubes. To each sample, 6 ml HPLC-grade methanol was added and sonicated in an ultrasonic water bath for 30 min before incubation at 80 °C for a maximum of 30 min. Samples were allowed to cool to room temperature and 1 ml of Milli-Q water was added, then vortexed at maximum speed for 1 min. Samples were centrifuged at 1 000 g for 1 min. The methanol layer was transferred to a clean tube and heated continuously in a 40 °C water bath until methanol had evaporated to completion. To each tube, 1 ml of HPLC-grade methanol was added and incubated at 40 °C for 15 min then filtered through 0.2 µm nylon membrane syringe filters (Chromatography Direct) into amber glass HPLC vials for later analysis. All chemicals and reagents were purchased from Thermo Fisher Scientific.

alkaline extraction (MeOH) protocol
A modified method of Caroll [15] was used for ergosterol extraction via MeOH. Of both growth substrate and root samples, 300 mg was weighed into 50 ml centrifuge tubes, as summarized in Fig. 1. Potassium hydroxide was added to HPLC-grade methanol until 10 % (w/v) was achieved. To each centrifuge tube, 10 ml of KOH in methanol was added and sonicated in an ultrasonic water bath for 15 min before incubation at 80 °C for a maximum of 30 min. Samples were allowed to cool to room temperature and 1 ml of Milli-Q water was added, then vortexed at maximum speed for 1 min. Samples were centrifuged at 1 000 g for 1 min. The methanol layer was transferred to a clean tube and heated continuously in a 40 °C water bath until methanol had evaporated to completion. To each tube, 1 ml of HPLC-grade methanol was added and incubated at 40 °C for 15 min then filtered through 0.2 µm nylon membrane syringe filters (Chromatography Direct) into amber glass HPLC vials for later analysis. All chemicals and reagents were purchased from Thermo Fisher Scientific.

Impact Statement
Ergosterol extractions typically employ methanol hydroxide as one of the main solvents. Inflated ergosterol quantification, and fungal biomass via equation transformation, can result from corrosive damage to plant cells. Therefore, alternative methods must be investigated. Furthermore, user exposure to hazardous chemicals, as well as overall length of the procedure, is greater than other solvent-based ergosterol extraction procedures carried out in the present study. Chloroform ergosterol extraction was seen to require the shortest period of user exposure to hazardous chemicals as well as the shortest extraction processing time. Chloroform extractions also were able to produce a greater quantity of ergosterol from the same samples as those subjected to methanol hydroxide extraction. This may suggest a chemical reaction between methanol hydroxide and ergosterol that reduces the quantity of the compound able to be extracted.

chloroform extraction protocol
A modified method of Alekseyeva et al. [18] was used for ergosterol extraction via methanol and chloroform. Of both growth substrate and root samples, 300 mg was weighed into 50 ml centrifuge tubes, as summarized in Fig. 1. To each sample, 3 ml of 2 : 1 chloroform to methanol was added and sonicated for 30 min at 50 °C in a closed tube. Samples were then allowed to cool to room temperature, followed by incubation at room temperature for 18 h. Samples were subsequently sonicated at 50 °C for 20 min and centrifuged at 1000 g for 1 min. Supernatant was transferred to a clean tube and heated continuously in a 40 °C water bath until methanol and chloroform had evaporated to completion. To each tube, 1 ml of HPLC-grade methanol was added and incubated at 40 °C for 15 min then filtered through 0.2 µm nylon membrane syringe filters (Chromatography Direct) into amber glass HPLC vials for later analysis. All chemicals and reagents were purchased from Thermo Fisher Scientific.

cyclohexane extraction protocol
A modified methodology originally developed by Millie-Lindblom et al. [9] was employed for the extraction of ergosterol [7]. Of both growth substrate and root samples, 300 mg was weighed into 50 ml centrifuge tubes, as summarized in Fig. 1. Potassium hydroxide was added to HPLC-grade methanol until 10 % (w/v) was achieved. To each centrifuge tube, 4 ml KOH in methanol and 1 ml cyclohexane was added and sonicated in an ultrasonic water bath for 15 min before incubation at 70 °C for a maximum of 2 h. Samples were cooled to room temperature and 1 ml of Milli-Q water was added with a further 4 ml cyclohexane, vortexed at maximum speed for 60 s then centrifuged at 1000 g for 60 s. The cyclohexane fraction was transferred to a clean test tube and all cyclohexane as evaporated, before 1 ml of HPLC-grade methanol was added and each tube incubated at 40 °C for 15 min then filtered through 0.2 µm nylon membrane syringe filters (Chromatography Direct) into amber glass HPLC vials for later analysis. All chemicals and reagents were purchased from Thermo Fisher Scientific.

Monitoring volatile exposure to the user
A ToxiRAE Pro PID (Honeywell) was kept by the user throughout the extraction procedure to monitor volatile organic compounds (VOCs) within the localized atmosphere to the user.

HPLc running protocol
The HPLC protocols were performed as described by Wilkes et al. [7], as a modified methodology of Mille-Lindblom et al. [9].

Fungal biomass estimation
Fungal biomass was determined from measured ergosterol concentration according to equation 1 [19]: where f is 250 and Rf (the recovery factor) is 1.61.

confirmation of aM fungi
Wheat and oak root sections were stained following the procedure of Wilkes et al. [20] for the visual confirmation of root intracellular AM fungal root structures.

Statistical analysis
Multivariate ANOVAs were used to determine if significant differences between sample/substrate type, plant species and ergosterol extraction produced an overall impact on the quantification of ergosterol. Single factor ANOVAs were performed between plant species of the same ergosterol extraction protocol, with post-hoc t testing, in order to determine the most effective ergosterol extraction procedure. Further t testing was caried out between ergosterol extraction protocols of the same plant species. Comparisons between ergosterol quantification of ECM and AM fungi were made via t testing. All data were analysed via R (version 4.1.0).
Construction of an ergosterol standard curve, via HPLC, indicated a retention time of 6.8 min (Fig. 3). Comparative analysis with root and growth substrate HPLC chromatographs (Fig. 4) presented ergosterol fractions of each extracted sample.   [7] and Millie-Lindblom et al. [9], indicating an ergosterol retention time of 6.8 min.
The total duration of the procedure is given in Table 1 along with the duration of exposure to hazardous chemicals for the respective extraction protocol. Use of a chloroform extraction protocol indicated significantly reduced hazard exposure time (P=0.005, d.f.: 2,9, F value: 4.04, F critical: 4.25, single-factor ANOVA). Due to the flow rate of the fume cupboard being used for all extraction procedures, monitoring of localized volatile hazards was consistently recorded at 0 ppm. Ergosterol was detected at 6.8 min (indicated by arrow) from comparison with a known ergosterol standard.

DIScuSSIOn anD cOncLuSIOn
The present comparison of methodologies has been able to show a large range in quantified ergosterol from the same samples by using four extraction procedures. Method comparisons indicated a consistently greater degree of extracted ergosterol, proportional to fungal biomass via equation 1, via a chloroform protocol compared to the other three extraction procedures used. MeOH extractions were seen to produce consistently low fungal biomass (Fig. 2) regardless of plant species, associated mycorrhiza or sample type. Fungal biomass, shown in Fig. 2, shows that wheat and oats maintain a higher fungal biomass on average, regardless of extraction protocol, compared to oak and roses. As explored by Wilkes [11] the use of MeOH in a cyclohexane ergosterol extraction procedure increases the quantity of ergosterol extracted from host root samples by damaging root cortical cells and exposing intracellular fungal membranes to the extraction procedure. ECM fungal biomass extracted from host roots can be seen from oak and roses in Fig. 2 and these showed reduced fungal biomass when compared with AM fungi-hosting wheat and oats. Whilst ECM fungi also have fungal mass within root systems of their host, in the form of intercellular hyphae forming the Hartig network [21], this was not seen to increase calculated fungal biomass (Fig. 2). A further potential explanation for the difference in quantified fungal biomass is the fibrous nature of oak and roses root tissue preventing ergosterol extraction from intercellular hyphae. This is not present to the same degree within wheat and oats as both of these crop types have very malleable root tissues that can be easily stained, dissected and used for extraction procedures without the need to add a further step to remove lignin in fibrous root tissues. However, equation 1 from Montgomery et al. [19] utilizes an ergosterol to fungal biomass conversion ratio. Whilst such a conversion can aid calculated percentage mass of fungi in a sample, sampled soils for example, the conversion factor assumes a constant ratio between ergosterol and the mass of fungal mycelia. This is unlikely to be constant between samples. It is for this reason that, especially in the study of mycorrhizal fungi, that a single quantified parameter is not sufficient to confirm the presence of the attribute being quantified. This is the justification for staining root samples, showing AM fungal root cortical structures, in Fig. 5. Montgomery et al. [19] reports the typical usage of ergosterol extraction for aquatic and ectomycorrhizal fungi. It is interesting to note that the quantified ergosterol in wheat and oat samples, both root and growth substrate, indicated high concentrations of ergosterol. However, as shown by Hart and Reader [22], AM fungi do not typically contain ergosterol. Therefore, it is likely that the quantified ergosterol present in wheat and oat samples is derived from saprophytic fungi.
The use of methanol alone for the extraction of ergosterol has been utilized by several investigations with a range of success [18, 23, 24]. The main difference in methanol alone to extract ergosterol is the duration of a heat treatment in a water bath or heating block dependent on the overall volume of the sample. Verma et al.
[25] placed samples in methanol in an 85 °C water bath for 30 min with hand shaking after 15 min followed by cooling to room temperature before filtering via a 0.22 µm syringe filter. This is similar to the methanol (non-alkaline) extraction procedure used in the present study, although the present study incubated the samples at 80 °C for 30 min with 30 min in an ultrasonic water bath. Verma et al. [25] found that a non-alkaline extraction procedure did not produce a greater concentration of ergosterol compared to alkaline methanol hydroxide protocols, although they did acknowledge that MeOH extractions did not allow finer soil matrices to be filtered out effectively and caused unreliable chromatographs upon HPLC analysis. This is not substantiated by the present study as an alkaline extraction procedure was able to produce the same chromatographic peak for ergosterol as methanol alone.
Typically, MeOH ergosterol extractions are coupled with cyclohexane to increase the concentration of the extracted ergosterol as seen in Fig. 2. Caroll [15] presents an MeOH ergosterol extraction without the addition of further solvents. The data presented by Caroll suggest ergosterol recovery rates ranged between 44 and 79 % for leaf samples. Such large ranges reduced the reliability of MeOH-extracted ergosterol values. From Fig. 2 of the present study, MeOH ergosterol recovery rates ranged between 0.6 and 24 % from plant root samples and between 1 and 75 % from growth substrate samples. Root samples support a greater abundance of fungal biomass compared to leaf samples. Leaf samples used by Caroll did not present with infection [15], a state that would have increased fungal biomass. The inflated values of ergosterol extracted by Caroll may be due to their standard curve being constructed from the peak height of HPLC chromatographs rather than peak area, which is proportional to the relative abundance of a corresponding molecule.
The employed chloroform/methanol ergosterol extraction procedure has several advantages over the other methods ued, including a single reaction tube, reduced equipment requirement, small volumes of solvent, and comparatively shorter It is acknowledged that all chemicals employed in the extraction of ergosterol are highly hazardous and pose a risk to the user, such as being corrosive and/or flammable. All extractions were carried out in a fume cupboard to reduce exposure to the user, as well as keeping a ToxiRAE Pro PID near the user to monitor volatile vapours released from the procedure. This was aided by keeping the lowest required volumes of each extractant chemical within the workspace of the fume cupboard. Such hazards, however, have to be balanced by the degree of reliably in extracting ergosterol from the sample. To this effect, the present comparison of methods would suggest the use of a chloroform extraction procedure for both root and growth substrate samples as this is also the procedure minimizing the overall duration of potential exposure to the user (Table 1). It is noteworthy that soils were not used to support the growth and development of the plant species used under controlled conditions. Therefore, the described procedures must be repeated on a wider range of sample types in order to broaden the application of the results demonstrated in the present study.

Funding information
This work received no financial support.
Author contributions T.I.W., conceptualization, method development, data generation, data analysis, manuscript writing, manuscript editing.  Reviewer 1 Comments to Author: The author has made substantial improvements to the manuscript and it is apparent chloroform is the most effective method for liberation and quantification of fungal biomass. However, the figures still need some review. In particular, presentation of statistical significance. Line 209: You do not need to include all elements of the statistical test, the P value is sufficient. Is this section simply indicating all methods were capable of liberating fungal biomass, but chloroform was most efficient?

Conflicts of interest
From previous experience, most journals and reviewers would prefer the full statistical description. In this case, I'm inclined to leave it in. Also, you are correct, this paragraph is to illustrate that all methods are capable of extracting ergosterol, however, as shown by the data, chloroform was by far the most effective method. An additional clarification has been added. Does your data simply imply chloroform proved significantly more effective than all other methods of extraction? Surely, there must be some variability in the P value between methanol hydroxide and cyclohexane relative to chloroform? R stats gave the increase in ergosterol quantification to ***P<0.0001. The actual P value was 2.3x10 -23 for all chloroform extractions regardless of the sample and species type. Additional details added.

Reviewer 2:
Please rate the manuscript for methodological rigour Please keep using words/abbreviations consistently throughout the manuscript such as ml or mL, minutes or min, 1 minute or 60s. Amended L209 Please explain briefly the details about multi-way ANOVA for overall results in the materials and methods to be related with the results here. It seems to be missing from the materials and methods.
Text added.
Amended Figure 1 Please capitalize the first letter in the flow chart boxes and correct "syringe" filter.
Amended Figure 2 Please check the number of samples. It should be n=300 instead of n=3,000 as running HPLC for 10 times per samples was not the real replicates. This is also concerned Table 1.

Recommendation: Minor Amendment
Comments: The author has made substantial improvements to the manuscript and it is apparent chloroform is the most effective method for liberation and quantification of fungal biomass. However, the figures still need some review. In particular, presentation of statistical significance. Figure 1: You could combine the two left methods at the point they overlap i.e. after the sonication and then have two arrows running down to a single flow chart. Results: Line 209: You do not need to include all elements of the statistical test, the P value is sufficient. Is this section simply indicating all methods were capable of liberating fungal biomass, but chloroform was most efficient? Figure 2: I am unsure of your presentation of statistical significance. Usually * means p

Please rate the quality of the presentation and structure of the manuscript Good
To what extent are the conclusions supported by the data? Strongly support

Anonymous.
Date report received: 21 November 2022 Recommendation: Accept Comments: I am satistfied with the responses from the author in amending the manuscript. However, there are some minor concerns as the followings. Please keep using words/abbreviations consistently throughout the manuscript such as ml or mL, minutes or min, 1 minute or 60s. In the results section, please update/correct the Figure number mentioned in the texts. L107 It confused the readers about how it ended up with n=300. Please simply clarify how many replicates of plant and growth substrate were sampled in the manuscript. L136 Is the verb e.g., "was added" missing? L148 Please change to "Alekseyeva et al. (2021)". L209 Please explain briefly the details about multi-way ANOVA for overall results in the materials and methods to be related with the results here. It seems to be missing from the materials and methods. L316 "height" instead of "hight". Figure 1 Please capitalize the first letter in the flow chart boxes and correct "syringe" filter. Figure 2 Please check the number of samples. It should be n=300 instead of n=3,000 as running HPLC for 10 times per samples was not the real replicates. This is also concerned Table 1.

Please rate the quality of the presentation and structure of the manuscript Good
To what extent are the conclusions supported by the data? Strongly support

If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
No: The manuscript does not involve any human and/or animal work. Some amendments have been made. However, from experience with several other papers, most journals and/or reviewers prefer trademarks.
As the same procedure was exploited to pre-prepare samples and the same volume of substrate used in each protocol (A total of 2g sampled rhizosphere growth substrate was air dried at 25°C for 48 117 hours and 300mg root material without drying. Of both growth substrate and root samples, 300mg was weighed into 50mL centrifuge tubes), this should only be described once in the initial methods section. This section should have a sub-heading labelled "sample preparation for extraction." Thank you for the suggestion. The text has been amended after noticing an error and moved into its own section Similarly the sentence assessing atmospheric VOCs should only be discussed once.

Amended
Results: Methods and statistical tests exploited should not be discussed in the results. Similarly, a p value is sufficient when discussing statistical differences. Significance between groups should be displayed on figures within discrete groups. For example, biomass liberated from oak. Please review publications and their presentation of significance. There are also a number of discussion points in the results which are not required.   Figure 2 is the known standard. I wouldn't typically combine that with samples. The software used for chromatograph generation doesn't allow the combination of chromatograms. I also think that some of the smaller peaks would be slightly more difficult to distinguish if they were on the same graph.

Discussion and conclusion
The discussion require review and should be shortened. The main focus of this section should be on the efficacy of chloroform in fungal biomass extraction and perhaps how this could be applied environmentally. For example, routine assessment of fungal growth and characterisation of the symbiotic relationship between plants and fungi.
The discussion has been amended, but has not been shortened. Aspects of the other extraction procedures must be discussed as well Ensure referencing is consistent throughout, in the introduction Vancouver is used, but in the discussion the same style is not exploited.
Could you please clarify? All references should be in Harvard format.
Leaf not leave Amended I'm not sure information regarding atmospheric VOC is necessary as you couldn't monitor this due to the use of a fume hood, I would suggest removing these data from your manuscript. This is true assuming the fume hood is properly maintained. The fume hood I used was recently serviced with a strong air flow. However, I have used fume hoods that did not have such a strong air flow and expose the user to the volatiles used in extraction procedures. The data was also included to satisfy health and safety as there were internal concerns regarding increased hazard from chloroform as opposed to methanol or methanol hydroxide.

Reviewer 2
Please rate the manuscript for methodological rigour

Reviewer 2: Good
Please rate the quality of the presentation and structure of the manuscript Reviewer 2 Comments to Author: 1. Methodological rigour, reproducibility and availability of underlying data Generally, the author clearly described the experimental design and the details of extraction methods used in the manuscript. This would benefit for reproducibility for further studies. All methods used employed ultrasonication and heat treatment to disrupt fungal and root cells, however, their conditions varied among the methods. Together with the ratio between the amount of sample and that of the solvent, this may cause differentiation of extraction ability and applicability, and thus should be concerned when interpreting and comparing the results. The use of ultrasonic bath could also lead to unequally energy obtained among the samples within each batch. This is a valid point and something I had considered early on in the study. However, the referenced publications justify the methods they report and the conditions of the extraction. Also, based on my previous work, methanol hydroxide extraction procedures don't require as much heat or sonication due to the corrosive nature of the solution. In these procedures, the plant tissues and cells are broken a part chemically rather than physically via sonication.
The underlying data were presented according to the statistical analysis with a number of samples (n=320). Moreover, monitoring the volatile vapours released during the extraction operation in the current study is very beneficial regarding health risks to the users besides the ergosterol yields or extraction ability.

Presentation of results
As the author performed ANOVA with post hoc test, would it be possible to include or present the statistic results on the result figure and table (such as on Figure 1 and in Table 1)? Added to figure   3. How the style and organization of the paper communicates and represents key findings The texts were concise, and structure of the overall manuscript was easy to follow. Using diagrams or table or restructuring the texts when describe the extraction methods in detail would help in reducing the redundant texts and being more visualizable to the readers. For example, describe only once about sample preparation as it was the same for all extraction methods. Furthermore, the extraction steps of all methods used were very similar to each other: extraction with organic solvent in association with cell disruption by ultrasonication and heat treatment, followed by liquid-liquid phase separation, then pre-concentration and purification prior to HPLC measurement. However, the conditions or details in every step differed across the methods. Thus, these may be presented as a comparison table for being more visualizable.
A summary flow chart has been included for each extraction procedure.

Literature analysis or discussion
The literature used in this manuscript sufficiently covered the underlying data and key findings. However, in accordance with Montgomery et al. (2000), the drawback of using fungal biomass estimation equation (Eq. 1) and its subsequent interpretation should be noted.
Added to lines 275 to 288

Any other relevant comments
Could the author explain briefly about ability or suitability of using ergosterol as a biomarker for AMF? I usually find that ergosterol is used to determine saprotrophic and/or ectomycorrhizal fungi in soils.
From my previous work, I have grown wheat in sterile conditions and inoculated the growth substrate with known AMF spores. Using Illumina sequencing to determine if contamination had been introduced (it hadn't), ergosterol was then quantified from the whet roots and growth substrate. Root staining had also been used to confirm the presence of AMF symbiosis. However, in the presently submitted study, no molecular investigation was carried out. Therefore, I have modified the text to take a more sceptical view on which type of fungi has been quantified from wheat and oats. However, the main focus of the manuscript is the extraction and quantification of ergosterol.
When or at which age were the samples collected? The materials and methods stated 6 months (L108) while the caption of Figure  1 was 9 months (L220).

Figure legend has been amended
Please check the number of samples as it was confusing among 20 (L101-102), 320 (L108) and 3,200 (L218). When n=320, does it mean that 16 samples of roots were collected from each plant together with 16 samples of their respective growth substrate?
Line 101 and 102 provide the plants used to generate the samples. In this respect, 20 plants of each species were used. Thank you for spotting n=320. There were 15 replicates of each sample extracted. This should have been n=300. These were then run through HPLC 10 times per sample, equalling 3,000 HPLC results to reduce retention errors within the HPLC system.
As compost was applied during the experiment, could it affect the ergosterol yield (probably from the saprotrophic fungi) particularly in growth substrate? Furthermore, were there any sample preparation for plant root tissue samples to avoid e.g., contamination of saprotrophic fungi?
No direct method was used to quantify saprophytic fungi. However, only 50g of compost was used in a larger volume of the perlite and vermiculite mix. Any quantified biomass of saprophytic fungi would have been fractional and unlikely to have had a large impact on the results displayed in the study. It is also acknowledged that working with complex microbiome samples, other methods of quantification for AMF should always be employed and correlated. There is a large body of literature that use several quantification processes in a single study to determine AMF abundance do to working with complex microbiomes. AMF can be quantified via stained root sections. This was performed for the cereal crops used, however, was not possible under the same procedure for ectomycorrhizal fungi.
Why the author used post hoc T testing and Bonferroni correction after ANOVA instead of other post hoc tests? Also, why performed (several) T test(s) among extraction protocols (4 extraction methods) within the same plant species instead of ANOVA? This may reduce the power of statistical test. What about using three-ways ANOVA?
A multi-way ANOVA was carried out initially to provide an overall glance at the differences between extraction protocols irrespective of specie. ANOVAs were used for individual species to further identify which extraction method more effective per species. Post hoc t tests were used to determine which extraction procedure had the greatest effect on the ANOVA significance.
Which statistical tests were performed for the volatile matter measurement data?
As all reading for volatiles were 0ppm, statistical tests did not provide any further results.
Please check the unit of the mean fungal biomass on Y-axis in Figure 1? Is it per g of roots or growth substrate? This is now figure 2. The units are derived from equation 1 and the figure also shows that the different coloured bars represent either root samples or growth substrate. An addition has been made to the figure legend to show that the results are from the extraction of 300mg of their respective sample.

Anonymous.
Date report received: 17 October 2022 Recommendation: Minor Amendment Comments: 1. Methodological rigour, reproducibility and availability of underlying data Generally, the author clearly described the experimental design and the details of extraction methods used in the manuscript. This would benefit for reproducibility for further studies. All methods used employed ultrasonication and heat treatment to disrupt fungal and root cells, however, their conditions varied among the methods. Together with the ratio between the amount of sample and that of the solvent, this may cause differentiation of extraction ability and applicability, and thus should be concerned when interpreting and comparing the results. The use of ultrasonic bath could also lead to unequally energy obtained among the samples within each batch. The underlying data were presented according to the statistical analysis with a number of samples (n=320). Moreover, monitoring the volatile vapours released during the extraction operation in the current study is very beneficial regarding health risks to the users besides the ergosterol yields or extraction ability. 2. Presentation of results As the author performed ANOVA with post hoc test, would it be possible to include or present the statistic results on the result figure and table (such as on Figure 1 and in Table 1)? 3. How the style and organization of the paper communicates and represents key findings The texts were concise, and structure of the overall manuscript was easy to follow. Using diagrams or table or restructuring the texts when describe the extraction methods in detail would help in reducing the redundant texts and being more visualizable to the readers. For example, describe only once about sample preparation as it was the same for all extraction methods. Furthermore, the extraction steps of all methods used were very similar to each other: extraction with organic solvent in association with cell disruption by ultrasonication and heat treatment, followed by liquid-liquid phase separation, then pre-concentration and purification prior to HPLC measurement. However, the conditions or details in every step differed across the methods. Thus, these may be presented as a comparison table for being more visualizable. 4. Literature analysis or discussion The literature used in this manuscript sufficiently covered the underlying data and key findings. However, in accordance with Montgomery et al. (2000), the drawback of using fungal biomass estimation equation (Eq. 1) and its subsequent interpretation should be noted. 5. Any other relevant comments Could the author explain briefly about ability or suitability of using ergosterol as a biomarker for AMF? I usually find that ergosterol is used to determine saprotrophic and/or ectomycorrhizal fungi in soils. When or at which age were the samples collected? The materials and methods stated 6 months (L108) while the caption of Figure 1 was 9 months (L220). Please check the number of samples as it was confusing among 20 (L101-102), 320 (L108) and 3,200 (L218). When n=320, does it mean that 16 samples of roots were collected from each plant together with 16 samples of their respective growth substrate? As compost was applied during the experiment, could it affect the ergosterol yield (probably from the saprotrophic fungi) particularly in growth substrate? Furthermore, were there any sample preparation for plant root tissue samples to avoid e.g., contamination of saprotrophic fungi? Why the author used post hoc T testing and Bonferroni correction after ANOVA instead of other post hoc tests? Also, why performed (several) T test(s) among extraction protocols (4 extraction methods) within the same plant species instead of ANOVA? This may reduce the power of statistical test. What about using three-ways ANOVA? Which statistical tests were performed for the volatile matter measurement data? Please check the unit of the mean fungal biomass on Y-axis in Figure  1? Is it per g of roots or growth substrate?